The respiratory epithelium provides a physical and an immunological barrier to inhaled pathogens. Pulmonary host defense mechanisms usually respond by entrapping microbes in the mucin layer and by releasing cytokines and chemokines. These signals alert alveolar macrophages and recruit neutrophils into the airways leading to elimination of the pathogen. In inhalation anthrax these defense mechanisms are subverted by B. anthracis to promote sporulation, bacterial growth and toxin secretion. Little is known about the effect ofB. anthracis and its toxins on lung epithelium function itself and on communication with innate immune cells. This project will investigate how B. anthracis infection alters lung epithelial cell responses. The influence of spores and bacilli components on signaling pathways in primary human airway cells will be studied. Air-liquid cultures will be used to determine if exposure to spores or bacterial products alters mucin and/or surfactant production. Airway epithelial cells internalize anthrax toxins leading to lethal factor-mediated proteolysis of signaling proteins. Our data reveal extensive morphological remodeling of the cytoskeleton and microtubule network in LeTx treated airway cells. The molecular mechanisms causing these changes and their influence on epithelial layer structure will be defined. Special emphasis will be on proteins involved in regulation of actin structures, microtubules or epithelial junctions such as Rho GTPases and their regulatory proteins. Additional LeTx targets in epithelial cells will be identified by proteomics. Airway epithelial cells contain NADPH oxidases (Nox/Duox), which are implicated in host defense and intracellular signaling. Nox/Duox-mediated generation of reactive oxygen species by B. anthracis components and/or toxins will be determined. Co-culture systems will be employed to determine if B. anthracis and its toxins alter the interaction between lung epithelial cells and immune cells. These studies will discern the impact of B. anthracis on the respiratory epithelium during anthrax infection and identify the underlying molecular mechanisms, a prerequisite for future therapeutic approaches.